Protein expression and purification of G-protein coupled receptor kinase 6 (GRK6), toward structure-based drug design and discovery for multiple myeloma

Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling,...

Full description

Saved in:

Bibliographic Details
Published in:	Protein expression and purification Vol. 185; p. 105890
Main Authors:	Olson, Tien L., Zhang, Shangji, Labban, Dillon, Kaschner, Emily, Aceves, Manuel, Iyer, Srivatsan, Meza-Aguilar, Jose Domingo, Zook, James D., Chun, Eugene, Craciunescu, Felicia M., Liu, Wei, Shi, Chang-Xin, Stewart, A. Keith, Hansen, Debra T., Meurice, Nathalie, Fromme, Petra
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-09-2021
Subjects:	Animals Antineoplastic Agents - chemical synthesis Baculoviridae - genetics Baculoviridae - metabolism Chromatography - methods Cloning, Molecular Crystallography Drug Design G-protein coupled receptor kinase 6 G-Protein-Coupled Receptor Kinases - chemistry G-Protein-Coupled Receptor Kinases - genetics G-Protein-Coupled Receptor Kinases - isolation & purification G-Protein-Coupled Receptor Kinases - metabolism Gene Expression Genetic Vectors - chemistry Genetic Vectors - metabolism Humans Multiple myeloma Multiple Myeloma - drug therapy Multiple Myeloma - enzymology Multiple Myeloma - genetics Neoplasm Proteins - chemistry Neoplasm Proteins - genetics Neoplasm Proteins - isolation & purification Neoplasm Proteins - metabolism Palmitoylation Protein Conformation Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Recombinant Fusion Proteins - metabolism Sf9 Cells Spodoptera Structure-guided drug design SEC CD Structure-guided drug design TEV IMAC Palmitoylation G-protein coupled receptor kinase 6 Multiple myeloma GRK6 DLS Crystallography
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and β-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma. •Three different constructs of G-protein coupled receptor kinase 6 (GRK6) were successfully expressed in insect cells.•Isolation and purification to homogeneity for biochemical characterization and crystallization achieved.•GRK6 folds properly in solution.•GRK6 actively binds to inhibitor.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1046-5928 1096-0279
DOI:	10.1016/j.pep.2021.105890